The formation of the peptide bond (peptidyltransferase) is a ribosomal activity which does not require soluble proteins or GTP, but which occurs when peptidyl-tRNA and aminoacyl-tRNA are bound at the proper sites on the ribosome. Previous results with 80 S ribosomes containing endogenous peptidyl-tRNA revealed that incubation with radioactive puromycin led to the incorporation of significant amounts of puromycin into a hot acid-insoluble form in the absence of EF-1 and EF-2 or GTP. The temperature-and time-dependent formation of peptidyl puromycin appeared to be catalyzed by a ribosomal activity; the reaction occurred with 0.5 M NH4Cl-washed ribosomes, although EF-1 and EF-2 activities seemed to be completely removed from the ribosomes by such treatment. More recent preliminary experiments with isolated, 60 S ribosomal subunits indicate that the reaction between exogenous acylaminoacyl-tRNA and puromycin, to yield acylaminoacyl-puromycin, can be readily carried out in the absence of 40 S subunits, soluble proteins, or GTP. The characteristics of the peptidyltransferase reaction with respect to the requirements for activity, optimal concentrations of reaction components and the mechanism of the peptide bond-synthesizing reaction will be carried out using highly purified, isolated 60 S subunits. Efforts will also be made to dissociate and reconstruct the particle, or to modify chemically various specific groups on 60 S proteins, and to determine the effects on peptidyltransferase.